Calcium ions play an important role in the central steps of blood coagulation. The emphasis of the current work is on understanding the role of metal ions in enzyme catalysis and/or protein complex formation in the blood coagulation cascade. We are employing lanthanide ions as substitutes for calcium ions and as probes of metal binding sites in bovine factors IX and X and bovine prothrombin. Using Eu(III) and Pr(III) as NMR shift reagents and Gd(III) as an NBR broadening reagent, we are studying the distance relationship between the metal ions and the gamma-carboxyglutamic acid residues in an N-terminal fragment of bovine prothrombin, residues (12-44). In additon, the relationship of the active site of activated factor X to high affinity metal binding sites is under study using fluorinated benzamidine derivatives. These studies utilize 19F NMR spectroscopy of the paramagnetic relaxation enhancement of the bound fluorine by bound lanthanide ions. An immunologic approach to exploring the role of metal ions in altering the three-dimensional structure of bovine prothrombin is currently being employed. These studies will utilize an operationally monospecific antibody to the N-terminal region of bovine prothrombin, residues (12-44), which has recently been isolated and characterized. BIBLIOGRAPHIC REFERENCES: Furie, B. and Furie, B.C.: Spectral changes in bovine factor X associated with activation by the venom coagulant protein of Vipera russelli. J. Biol. Chem., 251, 6807-6814, 1976. Furie, B., Greene, E., and Furie, B.C.: Metabolic fate of factor X in systemic amyloidosis associated with factor X deficiency. Blood, 48, 975, 1976.